Clinical and experimental observations suggest a role for gonadal hormones in modulation of the immune response. The most likely site of interaction between the immune and endocrine systems is the thymus gland. It has been long recognized that changes in androgen status are associated with changes in thymus size and we have demonstrated the presence of classic androgen receptors (AR) in thymocytes, suggesting that such changes are receptor-mediated. In previous studies, including work supported by this grant, we have used murine models to demonstrate effects of alterations in androgen status on the thymus. Androgen withdrawal and replacement and genetic syndromes of androgen resistance were found to be associated with changes in thymus size, composition and function. We now propose to continue to explore underlying cellular and biochemical mechanisms-with the following specific aims: 1. Identification of the cellular targets of androgen action in the thymus with examination of thymocyte and epithelial portions of the gland. 2. Determination of the role of transforming growth factor beta (TGF-beta) in androgen-mediated thymic involution. 3. Investigation of the role of the following physiologic processes in androgen-mediated thymic involution: A. Acceleration of programmed thymocyte death B. Mediation of thymocyte cell cycle entry Androgen status will be altered in normal male mice by castration with and without testosterone replacement. Cellular targets of androgen action will be identified at the RNA level using a nuclease protection assay for measurement of AR-specific RNA and the reverse transcription/polymerase chain reaction (RT/PCR) to identify AR in thymocytes and thymocyte subsets. Results will be confirmed at the protein level using specific anti-AR antibodies in immunohistochemical studies. Changes in TGF-beta levels with alterations in androgen status will be examined using nuclease protection and RT/PCR assays and confirmed using a specific ELISA and bioassays. Further assessment of whether TGF-beta recapitulates androgen effects will utilize intrathymic administration of TGF-beta. Effects of androgens on programmed cell death will examine DNA fragmentation and nuclease activation. The ability of androgens to alter thymocyte proliferation will be examined by measurement of DNA content, quantitation of cycling thymocytes using flow cytometry and histologic examination of thymus with bromodeoxyuridine and 3H thymidine labeling.